Plant Soil (2011) 338:399–409 DOI 10.1007/s11104-010-0553-5
REGULAR ARTICLE
Alterations in peroxidase activity and phenylpropanoid metabolism induced by Nacobbus aberrans Thorne and Allen, 1944 in chilli (Capsicum annuum L.) CM334 resistant to Phytophthora capsici Leo. Noé López-Martínez & Ma. Teresa Colinas-León & Cecilia B. Peña-Valdivia & Yolanda Salinas-Moreno & Patricia Fuentes-Montiel & Magdalena Biesaga & Emma Zavaleta-Mejía
Received: 14 July 2009 / Accepted: 23 August 2010 / Published online: 28 September 2010 # Springer Science+Business Media B.V. 2010
Y. Salinas-Moreno Campo Experimental Valle de México CIRCE INIFAP, Apartado postal 307, Km. 18.5 carr. Los Reyes-Lechería, Texcoco, Estado de México CP 56230, México
ammonia-lyase (PAL) activity, total soluble phenols (TSP) and chlorogenic acid concentration in CM334 plants inoculated with either or both pathogens (Na-Pc) were compared; also, the toxic effect of some phenolic acids on Na was tested in vitro. The highest POD activity (5.3 μM tetraguaiacol mg−1 protein min−1) was registered in plants inoculated only with Pc, while those inoculated only with Na showed the lowest (3.3 μM) (P≤0.05). PAL activity was 39.9 nM trans-cinnamic acid μg−1 protein min−1 in plants inoculated only with Pc, and it was lower (19.3 nM) and similar in noninoculated plants or those with Na and with Na-Pc (P≤0.05). Usually, plants inoculated with Pc alone had higher contents of TSP (P≤0.05) (1.9 mg tannic acid g−1 dry matter) and plants inoculated with Na or Na-Pc had lower levels (0.8 and 0.9 mg) than those noninoculated (1.3 mg). CM334 plants inoculated with Na showed a significant reduction (10–37% and 12–17%, in roots and leaves) in the concentration of chlorogenic acid as compared to the non-inoculated. Vanillic, transcinnamic, p-coumaric and syringic acids had greater nematicidal effects (P≤0.05) than chlorogenic acid in vitro. Apparently Na modified the defence responses in CM334 plants as POD and PAL activities and TSP and chlorogenic acid concentrations were reduced.
M. Biesaga Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
Keywords Chlorogenic acid . PAL . Peroxidase . Plant defences . Resistance breaking . Root-knot nematodes
Abstract Chilli CM334 (Capsicum annuum L.) is resistant to Phytophthora capsici Leonian (Pc), but Nacobbus aberrans Thorne and Allen, 1944 (Na) broke down its resistance in plants previously infected by the nematode. Peroxidase (POD) and L-phenylalanine Responsible Editor: Hans Lambers. N. López-Martínez : P. Fuentes-Montiel : E. Zavaleta-Mejía (*) Fitopatología, Colegio de Postgraduados, Km. 35.5 carr. México-Texcoco, Montecillo, Estado de México CP 56230, México e-mail:
[email protected] M. T. Colinas-León Departamento de Fitotecnia, Universidad Autónoma Chapingo, Km. 38.5 carr. México-Texcoco, Chapingo, Estado de México CP 56230, México C. B. Peña-Valdivia Botánica, Colegio de Postgraduados, Km. 35.5 carr. México-Texcoco, Montecillo, Estado de México CP 56230, México
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Introduction The chili pepper (Capsicum annuum L.) line of the “Serrano” type known as “Criollo de Morelos 334” (CM334) displays a high level of resistance to Phytophthora capsici Leonian and other phytopathogens as potyviruses and the three main species of root-knot nematodes [Meloidogyne arenaria (Neal) Chitwood, M. incognita (Kofoid and White) Chitwood and M. javanica (Treub) Chitwood] (Alcantara and Bosland 1994; Pegard et al. 2005); however CM334 is susceptible to Nacobbus aberrans Thorne and Allen, 1944. In previous studies we found that the CM334 chili pepper plants showed susceptibility when they were first inoculated with the false root knot nematode N. aberrans (Vargas et al. 1996; Trujillo-Viramontes et al. 2005; Godínez-Vidal et al. 2008). This phenomenon is known as “resistance breaking” (Sasser et al. 1955; Hernández et al. 1992; Maheshwari et al. 1995). The breakdown of resistance in CM334 plants took place when a minimum of 300,000 zoospores and 2,000 second stage juveniles (J2) per plant were inoculated, and the maximum degree of resistance breakdown of chilli CM334 occurred when P. capsici was inoculated 21 days after having inoculating the plants with N. aberrans (Trujillo-Viramontes et al. 2005). In some chili pepper varieties, the resistance to P. capsici is associated with L-phenylalanine ammonialyase (EC 4.1.3.5; PAL) activity. PAL is the key enzyme of the phenylpropanoid pathway. Branches of this pathway lead to the synthesis of compounds that have diverse functions in plants, such as those related to defence, i.e. lignin, suberin, phenolic compounds, coumarins and isoflavonoid phytoalexins, and signaling compounds such as salicylic acid (Shadle et al. 2003; Gómez-Vásquez et al. 2004). It has been reported that PAL genes, which code for PAL synthesis, are down-regulated by the root-knot nematode Meloidogyne incognita in compatible plantpathogen interactions (Goddijn et al. 1993). In contrast, M. incognita stimulates hmg gene expression, which codes for hydroxymethyl-glutaryl-CoA reductase (EC 1.1.1.34; HMGR). This enzyme plays a critical role in sterol biosynthesis (Benveniste 2004). It is important to emphasize that the phytoparasitic nematodes are unable to synthesize sterols and are therefore completely dependent on their host to obtain
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them (Chitwood and Lusby 1991). It is also known that sterols are important for growth and sexual and asexual reproduction of Phytophthora spp., including P. capsici (Elliot 1983). Peroxidase (E.C. 1.11.1.7; PO) has also been involved in resistance mechanisms to pathogens. An increase in peroxidase activity was associated with the CM334 resistance to P. capsici (Fernández-Pavia 1997) as compare to susceptible plants. As a resistance mechanism to pathogen attack, this enzyme thickens cell walls and generates active oxygen species (Hiraga et al. 2001; Anterola and Lewis 2002). In incompatible plant-nematode interaction, peroxidases play a critical role in the generation of reactive oxygen species (ROS) associated with hypersensitive reaction (HR) (Melillo et al. 2006). It has been reported that acumulation of phenolic compounds in chili pepper CM334 might be involved in the resistance to different species of genus Meloidogyne (Pegard et al. 2005). Indeed, an important increase of chlorogenic acid was associated with the resistance of CM334 to root-knot nematodes. It is thought that the accumulation of this acid could be responsible for the browning and the resistant reaction of both Nemared tomato cv and CM334 chilli pepper to M. incognita (Huang and Rhode 1973; Pegard et al. 2005). The oxidation of phenolic compounds could be due to enzymatic activity of peroxidases or polyphenol oxidases (Gómez-Vásquez et al. 2004). Chan, cited by Huang and Rhode (1973), reported that the oxidation products of chlorogenic acid reduced the respiration of Pratylenchus penetrans (Cobb 1917) Filipjev and Schyuurmans-Stekhoven 1941. Since CM334 is susceptible to N. aberrans it would be interesting to know whether this nematode is insensitive to the toxic effect of chlorogenic acid or is able to inactivate it, or if the nematode suppresses the biosynthesis of this compound. On the other hand, among phenolics that inhibit mycelial growth of P. capsici, trans-cinnamic acid stands out for its inhibitory effect (Candela et al. 1995). Based on the above, we tested the hypothesis that infection by N. aberrans reduces some defence responses to P. capsici in chilli CM334. Therefore, the peroxidase and L-phenylalanine ammonia-lyase activities and total soluble phenols were compared in CM334 plants inoculated with either and both pathogens. In addition, a comparison of the concen-
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tration of chlorogenic acid in P. capsici resistant (line CM334) and susceptible (cv. J. E. Parker) plants inoculated and non-inoculated with N. aberrans or M. incognita was carried out, and the toxic effects of some commercial phenolic acids on the nematode were tested in vitro.
Materials and methods Chili pepper plants Seeds of resistant (CM334) and susceptible (J.E. Parker) chilli genotypes were surface-sterilized by immersion in a 1% sodium hypochlorite solution for 1 min, and rinsed in sterile distilled water. The seeds were germinated at 28±1°C on filter paper in Petri dishes. After germination, the seedlings were transplanted singly into pots containing 150 mL of sterile sand. The plants were irrigated daily with sterile distilled water, and fertilized three times a week with Nitrofoska® (12-12-12-2; N-P-K-Mg): 620 g of Nitrofoska were dissolved in 20 L of water, and then diluted in 10 parts of water. The pots were maintained in growth chambers at 28±1°C, 70 to 80% relative humidity and with a 14-hour photoperiod at a luminous intensity of 6,768 lux (fluorescent light) and 10 h of dark.
Inoculum preparation and inoculation Nematodes Egg masses were extracted from roots of tomato plants infected with M. incognita or N. aberrans. The egg masses were incubated at 28°C in Petri dishes containing the fungicide Captan® (0.1%) and the antibiotic Chloramphenicol® (0.1%) in sterile distilled water until they hatched. Hatched second stage juveniles (J2) were counted and a suspension containing 200 J2 per mL was prepared. Each plant was inoculated by pouring 10 mL of the suspension (containing 2,000 J2) when plants had grown four to five leaves. Phytophthora capsici Inoculum production and inoculation were carried out according to TrujilloViramontes et al. (2005). The isolate 6143 of P. capsici was provided by Dr. Sylvia Fernández-Pavia.
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Discs of PDA (0.5 cm diameter) with mycelium of the oomycete were placed in the centres of Petri dishes containing V8 medium (355 mL of V8 juice, 4 g of CaCO3, 16 g of agar, in 1 L) and then incubated at 28°C in darkness. Twenty days later, the medium was divided into four segments and each was transferred to another Petri dish containing 20 mL of sterile distilled water to induce formation of sporangia. Petri dishes were maintained at 28°C for 10 days. Induction of zoospore liberation was carried out by exposing the mycelia to 4°C for 2 h. The number of zoospores was estimated using a haemocytometer (Marienfeld ®). Each plant was inoculated with 3×105 zoospores of P. capsici. The pots were saturated with sterile distilled water 24 h before inoculation. In experiments involving the oomycete, plants of the cv. J.E. Parker were also inoculated as a reference for its pathogenicity and inoculation effectiveness.
Experiments establishment Experiment 1 In this experiment, the root peroxidase and PAL activities, and total soluble phenols were determined after 6 h of P. capsici inoculation in 1) non-inoculated CM334 plants (Control), 2) CM334 plants inoculated only with N. aberrans (Na), 3) CM334 plants inoculated with P. capsici alone (Pc), and 4) CM334 plants inoculated with both pathogens (Na-Pc). Inoculation with the oomycete was carried out 21 days after inoculation with the nematode. Between 12 and 20 backup plants were used in each case. At 6 h after inoculation, roots were removed from the plants and washed in running tap water. The roots were pooled and immediately frozen in liquid nitrogen. A composite sample from each of the four treatments was broken into small pieces and stored at −80°C until analysis. The root tissue from each treatment was blended separately with chilled acetone (1:10, w:v) and filtered. The filtrate was used for determing total soluble phenols. The solid residue was washed five times with chilled acetone and then dried at room temperature. The resulting powder (acetone powder) was stored at −20°C until used for enzyme activity determination. During the extraction procedure the samples were kept in ice. The experiment was repeated once.
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Experiment 2 This experiment aimed to identify and quantify the chlorogenic acid content in the resistant genotype CM334 (R) and the cv J.E. Parker, susceptible (S) to both P. capsici and N. aberrans. The treatments were: 1) resistant plants with Na (R Na), 2) resistant plants without Na (R), 3) susceptible plants with Na (S Na), and 4) susceptible plants without Na (S). There were five plants per treatment and each was inoculated with 2,000 Na J2 at the stage when they bore four to five leaves. Five days after inoculation, roots were harvested and carefully washed with running tap water. From the plants of each treatment segments of leaves and roots (0.4 g each) were collected, and placed separately in 10 mL of 100% methanol for 24 h. The obtained extracts were used for chlorogenic acid identification and quantification; quantification was done in five replications. The experiment was replicated once, but in this case the incompatible interaction Meloidogyne incognitaCM334 was also included, therefore there were six treatments: 1) CM334 plants inoculated with Na (R Na), 2) CM334 plants inoculated with M. incognita (R Mi), 3) CM334 plants not inoculated (R Control), 4) J.E. Parker plants inoculated with Na (S Na), 5) J.E. Parker plants inoculated with Mi (S Mi), and 6) J.E. Parker plants not inoculated (S Control). The inoculation, sampling and chlorogenic acid extraction were carried out as indicated above.
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dish, which allowed live nematodes to migrate downwards through the filter paper into the Petri dish. The J2 were counted and percentage mortality was calculated according to Mahajan et al. (1985), using the formula ½½ðB AÞ=B 100; where B is the number of recovered nematodes in the control and A the number of nematodes recovered from the treatment. This test was repeated three times.
Protein extraction and peroxidase activity These assays were performed at 4°C. Five replications of 25 mg of the acetone powder were homogenized in 5 mL of buffer [50 mM Tris–HCl (pH 7.1) and PVP 1% (w:v)] in an Ultra Turrax blender (20,000 rpm for 1 min). The homogenate was filtered through four layers of cotton mesh, and centrifuged at 13,000 g for 20 min. The peroxidase activity was quantified in the supernatant. Protein concentration was determined by the Bradford method (1976), using bovine serum (Sigma Aldrich) as standard. Peroxidase activity was determined as described in Chance and Maehly (1955). Enzyme activity was expressed as μM tetraguaiacol mg−1 protein min−1. For Peroxidase activity there were two independent experiments.
Protein extraction and PAL activity
Nematicidal activity of phenolic compounds Second stage juveniles of N. aberrans were extracted as previously described. Two mL from a suspension containing approximately 100 J2 per ml of water were poured into Petri dishes (diameter 40×15 mm height) containing 2 mL of an aqueous solution of transcinnamic acid (Aldrich 99%), vanillic acid (Fluka ≥ 97%), chlorogenic acid (Sigma 95%), p-coumaric acid (Sigma 100%), syringic acid (Sigma, minimum 98%), ferulic acid (Sigma), or tannic acid (Sigma Aldrich), obtaining 1 mg mL−1 as final concentration of each phenolic acid. A control with distilled water was included. The Petri dishes were maintained in the dark at room temperature for 48 h. After this, juveniles were recovered on a plastic mesh supporting a coarse filter paper in contact with water contained in a small Petri
Five replications of 25 mg of acetone powder were homogenized in 5 mL of 0.1 M borate buffer (pH 8.8) with 1% PVP (w/v) and 0.02 M β-mercaptoethanol in an Ultra Turrax blender (20,000 rpm for 1 min). The homogenate was filtered through four cotton mesh layers, and centrifuged at 13,000 g for 20 min. The extract was purified by salting out proteins with ammonium sulphate to a final saturation of 46% and centrifuged at 13,000 g for 20 min. The precipitated protein was dissolved in 4.5 mL of 0.1 M ammonium acetate buffer, pH 7.7, containing 0.02 M βmercaptoethanol. Protein concentration was determined as described above. Activity of PAL was determined in accordance to Martinez-Tellez and Lafuente (1997), by measuring the production of trans-cinnamic acid from L-phenylalanine spectrophotometrically at 290 nm, and using a transcinnamic acid standard curve; PAL activity was
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expressed as nM trans-cinnamic acid μg−1 protein min−1. Data for PAL activity were obtained from two independent experiments.
Quantification of total soluble phenols (TSP) The TSP of each extract was determined using the method previously described by Waterman and Mole (1994). The appropriate dilutions of extracts (in each 600 μL of acetone root extraction, deionized water was added to make a final volume of 17 mL) were oxidized with Folin-Ciocalteu reagent (Sigma Aldrich), and the reaction was neutralized with sodium carbonate (10% w/v). The absorbance of the resulting blue colour was measured at 760 nm after 2 h, using tannic acid as standard (Sigma Aldrich), TSP was expressed as mg−1 g of tannic acid g−1 dry matter. For TSP two independent experiments were conducted each with five replicates.
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linearity was determined from squared correlation coefficient of the calibration curve generated by three repeated injections of standard solutions at five concentration levels (1–50 mg/L). All data were reported as means of five replicates. All solvents were of chromatographic grade (Sigma Aldrich).
Statistical analysis The experiments were conducted in a completely random design. Unless otherwise indicated, data from replicates of the same experiment were pulled together for a single ANOVA, and differences between means of treatments were determined using Tukey’s test. Statistical procedures were performed using Statistical Analysis System software for PC (SAS Institute 1999–2000, version 8.1); differences with P≤0.05 were considered significant.
Results Identification and quantification of chlorogenic acid Identification and quantification of chlorogenic acid was carried out by High-Performance Liquid Chromatography (HPLC), as described by Pegard et al. (2005). Twenty-four hours after having placed plant tissue in methanol, the extract was dried under vacuum in a rotary evaporator at 38°C. The concentrated extract was solubilised in 1 mL of methanol:acetic acid:water (25:1:75 v/v/v) solution, filtered (0.45 μm), and stored at −17°C until analyzed. The separations were carried out on a HPLC system from Perkin Elmer Series 200, equipped with DAD detector. The analytical column was Hypersil ODS.2 (250×4,6 mm, particle size 5 μm) set at a temperature of 30°C. The analytical conditions consisted of a isocratic elution using a mixture of methanol:acetic acid:water (25:1:75, v/v/v), with a flow rate of 1 ml/min, and a running time of 20 min. The amount of sample injected was 20 μL. The data were collected and evaluated by Perkin-Elmer chromatographic software TotalChrom 200. Identification of chlorogenic acid in extracts was based on comparison of retention time of a commercial standard (Sigma, MN) and the spectrum at 254 nm. We prepared a standard curve of chlorogenic acid for quantifying this phenolic acid in the extracts. The
Peroxidase and PAL enzymatic activities and total soluble phenols Peroxidase activity There were significant differences (P≤0.05) in peroxidase activity in all treatments (Table 1). The activity in resistant CM334 pepper chilli plants inoculated only with the oomycete was increased by 23% as compared to the control plants. In contrast, the activity of peroxidase was decreased by 18% and 24% in plants inoculated with the nematode alone and in plants where both pathogens were present, respectively. PAL activity The activity of the enzyme was significantly (P≤0.05) different among treatments (Table 1). In the incompatible interaction (CM334 plants-P. capsici), the activity was increased by 45% in comparison with non-inoculated control plants at 6 h after oomycete inoculation; however, PAL activity was lower than control plants when N. aberrans was present alone or in combination with the oomycete, with a 30% and 26% reduction, respectively. Total soluble phenols (TSP) The content of TSP in pepper chilli plants from the different treatments
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Table 11 Enzyme Enzyme(peroxidase (peroxidaseandand PAL) PAL) activities activities and total and soluble total phenols (Pc), or(TSP) both in pathogens chilli pepper (Na-Pc) plants at (Capsicum 6 h after inoculation annuum) CM334 with P. soluble inoculatedphenols with N. (TSP) aberrans in (Na), chilliP. pepper capsici (Pc), plantsor (Capsicum both pathogenscapsici. (Na-Pc)The at 6oomycete h after inoculation inoculationwith was P.carried capsici. outThe 21 days oomycete after annuum) inoculationCM334 was carried inoculated out 21with daysN.after aberrans nematode (Na),inoculation P. capsici nematode inoculation Treatment
Peroxidase (μM tetraguaiacol mg−1 protein min−1)
PAL (nM trans-cinnamic acid μg−1 protein min−1)
TSP (mg tannic acid g−1 dry matter)
4.3
B
27.4
B
1.3
B
Pc
5.3
A
39.9
A
1.9
A
Na
3.3
C
19.3
C
0.8
C
3.5
C
20.4
C
0.9
C
Control
Na-Pc C.V.
12.1
18.1
1.9
Values are the means of 10 replications from two independent experiments. In each column, values followed by the same letter are not significantly different according to Tuke’s test (P≤0.05, Tukey’s test). C.V. = Coefficient of variance
followed the same pattern as peroxidase and PAL activities (Table 1). When P. capsici was inoculated alone, the TSP content was significantly (P≤0.05) increased by 46% in comparison with control plants. The opposite was observed when the plants were inoculated with N. aberrans alone or in combination with P. capsici; the TSP were reduced by 38% and 31%, respectively.
Chlorogenic acid content in plants inoculated with N. aberrans Extracts from CM334 and J.E. Parker chilli plants inoculated and non-inoculated with N. aberrans showed peaks with a retention time (6 min) identical to the standard chlorogenic acid used. The content of this phenol at 5 days after nematode inoculation was higher in resistant plants than in the susceptible ones (P≤0.05). In contrast, the content of chlorogenic acid was significantly lower in roots and leaves of susceptible (J. E. Parker) and resistant (CM334) plants inoculated with the nematode as compared to the non-inoculated (Table 2). The leaves of CM334 had a higher concentration of chlorogenic acid (more than twofold) than those from J.E. Parker. In both genotypes, the leaves showed higher concentrations than roots (5.9- and 3.4-fold in resistant and susceptible plants, respectively). The level of chlorogenic acid in CM334 roots inoculated with N. aberrans was 10% lower than noninoculated plants, while in roots from susceptible plants inoculated with the nematode the level was 17% lower than in non-inoculated plants. Chlorogenic acid content in leaves was also significantly (P
